Design and discovery of novel thiazole derivatives as potential MMP inhibitors to protect against acute lung injury in sepsis rats: Via attenuation of inflammation and apoptotic oxidative stress

Article abstract of DOI:10.1039/c7ra03511j

Acute lung injury (ALI) is considered to be an inflammatory syndrome of the airway system that is initiated by failure of the respiratory system. In this study, we evaluated the possible benefits of some novel thiazole derivatives against ALI. These derivatives were synthesised and evaluated for the inhibition of MMP-8 and MMP-2. Most of the tested compounds had better inhibitory activity for MMP-8 than for MMP-2, with compound 26 being the most potent analogue among the tested series. Thus, compound 26 was further investigated to determine its beneficial effects in an ALI model of rats with sepsis. In vivo results suggested that compound 26 significantly reduced the protein concentration together with a decline in enhanced leukocytes compared with those in ALI induced by cecal ligation and puncture. The effect of compound 26 on myeloperoxidase activity was also quantified, which was found to be significantly reduced at the maximum tested dose of 20 mg kg^-1. The protective effect of compound 26 against ALI was also established to occur via the significant modulation of various biomarkers; for example, the malondialdehyde level was found to be reduced, while there were increased levels of superoxide dismutase and glutathione. Thus, it is proposed that compound 26 exerts a protective effect against ALI via modulation of the antioxidant status. Furthermore, the compounds tested caused significant attenuation of the levels of tumour necrosis factor-α, interleukin-1β, and interleukin-6, and protected the lung through the modulation of systemic inflammatory mediators in septic rats. In conclusion, we identified a novel series of thiazoles, which potentially exert protective effects against ALI via the inhibition of numerous pathways.

Full text of DOI:10.1039/c7ra03511j

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PAPER
Design and discovery of novel thiazole derivatives
as potential MMP inhibitors to protect against acute
lung injury in sepsis rats via attenuation of
inflammation and apoptotic oxidative stress
c
Cite this: RSC Adv., 2017, 7, 32909
Lingqing Ge,a Qiaozhen Hu,b Mengrao Shi,a Huiyun Yanga and Guoji Zhu
*
Acute lung injury (ALI) is considered to be an inflammatory syndrome of the airway system that is initiated by
failure of the respiratory system. In this study, we evaluated the possible benefits of some novel thiazole
derivatives against ALI. These derivatives were synthesised and evaluated for the inhibition of MMP-8 and
MMP-2. Most of the tested compounds had better inhibitory activity for MMP-8 than for MMP-2, with
compound 26 being the most potent analogue among the tested series. Thus, compound 26 was further
investigated to determine its beneficial effects in an ALI model of rats with sepsis. In vivo results suggested
that compound 26 significantly reduced the protein concentration together with a decline in enhanced
leukocytes compared with those in ALI induced by cecal ligation and puncture. The effect of compound 26
on myeloperoxidase activity was also quantified, which was found to be significantly reduced at the
maximum tested dose of 20 mg kgꢀ1. The protective effect of compound 26 against ALI was also
established to occur via the significant modulation of various biomarkers; for example, the malondialdehyde
level was found to be reduced, while there were increased levels of superoxide dismutase and glutathione.
Thus, it is proposed that compound 26 exerts a protective effect against ALI via modulation of the
antioxidant status. Furthermore, the compounds tested caused significant attenuation of the levels of
tumour necrosis factor-a, interleukin-1b, and interleukin-6, and protected the lung through the modulation
of systemic inflammatory mediators in septic rats. In conclusion, we identified a novel series of thiazoles,
which potentially exert protective effects against ALI via the inhibition of numerous pathways.
Received 26th March 2017
Accepted 7th June 2017
DOI: 10.1039/c7ra03511j
rsc.li/rsc-advances
severe form, acute respiratory distress syndrome (ARDS).10 It is
considered to be an inammatory syndrome of the airway
Introduction
system initiated by failure of the respiratory system, resulting
from direct/indirect damage to the vasculature of the lung.11,12
This damage leads to increased permeability of leukocytes,
together with an enhanced inammatory response and oxida-
tive stress in the lungs.13,14 Novel agents rather than just venti-
lation are urgently needed to treat sepsis-induced ALI/ARDS and
attenuate the associated pathological changes. Because of the
involvement of septic shock in multi-organ failure,15 various
studies have focused on the management and attenuation of
the inammatory response, which is thought to be critically
involved in the induction of sepsis.16,17 It has also been
conrmed that metalloproteinases (MMPs) play a key role in the
regulation of the septic inammatory response.18,19 Apart from
degradation of the extracellular matrix (ECM), MMPs are
thought to be responsible for modifying the diverse group of
non-ECM proteins, which ultimately interfere with the inam-
matory process.20 Various rodent studies of sepsis have
conrmed that the inhibition of MMPs has a signicant inu-
ence on the expression of inammatory markers, together with
improved levels of mortality in the experimental animals.21
Sepsis is classied as a general inammatory response induced
by infection during burns, trauma, or hypoxia, and aer
surgery, which oen leads to multi-organ dysfunction and
death.1–3 Despite recent advances in the modalities of clinical
approaches for the management of sepsis, there are still diffi-
culties in managing patients with this condition.4,5 Various
studies and statistical analyses have revealed that, each year,
more than 18 million people suffer from sepsis.6 The number of
septic infections is rising at an annual rate of 1.5–8.0%, with
mortality ranging from 25% to 50%.7,8 Among cases of sepsis,
the lung is classied as the most affected organ and lung
damage is largely responsible for the resulting mortality.9 Lung
function is greatly hindered by acute lung injury (ALI) and its
aNeonate Department, Soochow University Affiliated Children's Hospital, Suzhou,
Jiangsu 215003, P. R. China
bObstetrical Department, Suzhou Hospital of Traditional Chinese Medicine, Suzhou,
Jiangsu 215003, P. R. China
cDepartment of Internal Medicine, Soochow University Affiliated Children's Hospital,
Suzhou, Jiangsu 215003, P. R. China. E-mail: zhuguoji189@hotmail.com; Fax: +86-
512-6522-3820; Tel: +86-512-6522-3820
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The non-selective inhibition of MMPs oen leads to an bands at 3398–3387 cmꢀ1, which conrmed the presence of
irrelevant pharmacological response because of its involvement a secondary amine group. There was another strong band at
in many regulatory processes.22 Thus, the selective targeting of 3143–3142 cmꢀ1, which was attributed to stretching vibrations
specic MMPs can overcome the unwanted side effects.23 of the C–H group of the aromatic ring. Another strong band at
Among the family of MMPs, MMP-8, a neutrophil-derived 1198–1187 cmꢀ1 conrmed the presence of an aromatic F
collagenase, is believed to be involved in damage to type I group. The thiazole C–H group appeared at 2859–2848 cmꢀ1
.
collagen.24 It has also been shown to be present in a diverse Another strong band at 1712–1723 cmꢀ1 conrmed the pres-
range of cell types, such as macrophages, broblasts, epithelial ence of the CO group. The aromatic chloro group appeared at
cells, and other immune cells, and can regulate pro- 798–786 cmꢀ1. The strong band at 662–647 cmꢀ1 was attributed
inammatory chemokines.25 Among the heterocyclic to stretching vibrations of the C–S group of the thiazole ring.
compounds, the thiazoles and their derivatives are important The 1H NMR spectra of the synthesised derivatives 6–30
bioactive molecules due to their excellent pharmacological revealed a doublet corresponding to the aromatic ring at 8.15–
activity.26 The signicance of thiazoles is emphasised by the fact 6.61 ppm. Furthermore, the resonance at 7.58–7.51 ppm
that various drugs originate from them, such as antineoplastic conrmed the presence of a phenyl thiazole proton as a singlet
agents (tiazofurin and dasatinib),27 an anti-HIV drug (ritona- peak. The side chain methylene proton appeared at 3.19–
vir),28 an antifungal agent (ravuconazole),29 an antiparasitic 3.14 ppm as a single peak. The phenyl thiazole side chain NH
agent (nitazoxanide),30 and an antiulcer agent (nizatidine).31 proton appeared at 9.16–9.13 as a singlet peak. Furthermore,
Thiazoles have also been reported to exhibit anti-inammatory the resonance at 3.42–2.46 ppm conrmed the presence of
activity32 and are included among clinically relevant drugs, such a methylene proton, with a doublet peak of the piperazine ring.
as fanetizole, meloxicam, and fentiazac.33 Ozdemir et al. re- Finally, the structure of all of the synthesised derivatives 6–30
¨
ported the MMP inhibitory activity of some thiazoles.34 Thus, in was conrmed by mass spectrometry and elemental analysis.
this study, we attempted to determine the effect of some novel
Table 1 presents the MMP-2 and MMP-8 inhibitory activities
thiazole derivatives against MMP-8 and subsequently investi- of the designed analogues in the form of comparative inhibitory
gated their benecial effects in an ALI model of rats with sepsis. proles determined via residual enzyme activity, by means of
continuous uorometric assays in the presence of the uores-
cent substrate QF 24. The results suggest that the majority of
molecules had a considerable inhibitory prole, with some
Result
Chemistry
exceptions where molecules showed less or no activity. In
particular, molecules developed in step 1 displayed no inhibi-
tory activity, whereas the introduction of an extended
substituted side chain resulted in an improvement in activity,
with the exception of compounds 6–13. The introduction of
chloro and uoro groups resulted in compounds being
considerably more active against both MMP-2 and MMP-9.
Compounds containing electron-donating substituents (17
and 18) showed mild to moderate activity, with no inhibition in
the case of compound 16. The presence of an electron with-
drawing group in the R2 position resulted in enhanced activity.
The next series of compounds (21–30) showed excellent inhib-
itory activity compared to other analogues, which could be
easily understood by the presence of an electron withdrawing
group. The comparison of inhibitory activity indicated that
molecules containing an electron withdrawing group at both
sites exhibited more activity than non-halogen congeners. From
The title compounds were synthesised with an excellent yield
via the facile synthetic route shown in Schemes 1 and 2. In the
rst stage, various substituted phenyl thiazoles 3(a–e) were
obtained by the reaction of acetophenone 1(a–e) with thiourea
(2) using ammonia solution as a base. In the second stage, 2-
chloro-N-[4-(p-substituted phenyl)-thiazol-2-yl]-acetamides 4(a–
e) were obtained by the reaction of substituted phenyl thiazole
3(a–e) with chloroacetyl chloride, as shown in Scheme 1. Finally,
the title compounds 6–30 were obtained by the reaction of 2-
chloro-N-[4-(p-substituted phenyl)-thiazol-2-yl]-acetamides 4(a–
e) with various phenyl piperazine derivatives 5(i–v) in ethanol,
as shown in Scheme 2.
The structures of all synthesised compounds were ascer-
tained by mass spectrometry, 1H-NMR, 12C-NMR, FT-IR, and
elemental analysis. The FT-IR spectra of all synthesised deriv-
atives 6–30 were characterised by the appearance of strong
Scheme 1 Reagents and condition: (a) Br2, reflux 8 h (b) chloroacetyl chloride, reflux 6 h.
32910 | RSC Adv., 2017, 7, 32909–32922
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Scheme 2 Reagents and condition: (A) ethanol, reflux 9 h.
Table 1 In vitro inhibition of MMP-2 and MMP-8 by target compounds
IC50 (in mM)
inhibitory activity of compound 26, we then explored the effects
of this compound on the in vivo sepsis-induced lung injury of
rats. As shown in Fig. 1A, it was found that compound 26
resulted in improvements following the histopathological
examination of the lung tissues of experimental animals
exposed to different treatments aer staining with H&E. The
sham-treated group showed no abnormal architecture, whereas
the cecal ligation and puncture (CLP) group had an abnormal
micro-structure, with disruption to the vasculature system,
increased permeability of inammatory cells into the alveolar
sac, and damage to the alveolar structure. These changes were
signicantly reversed to normal in the treatment group
Compound
R1
R2
MMP-2
MMP-8
4a
4b
4c
4d
4e
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
H
—
—
—
—
—
H
4-CH3
4-OCH3
4-Cl
4-F
ND
ND
ND
ND
ND
ND
ND
ND
4-CH3
4-OCH3
4-Cl
4-F
H
H
H
H
H
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-Cl
ND
ND
>100
>100
>100
>100
>100
>100
>100
>100
34.31
32.66
>100
24.40
23.28
18.52
18.04
14.34
16.73
15.31
14.34
13.25
4.22
>100
>100
>100
>100
>100
>100
>100
>100
29.23
35.05
>100
23.37
21.45
19.62
18.21
16.27
14.44
13.02
11.95
11.03
2.34
receiving compound 26 at the maximum dose of 20 mg kgꢀ1
.
The weight ratio of wet against dry lungs was estimated to
conrm the effect of compound 26 on pulmonary oedema, the
results of which are presented in Fig. 2B. It was observed that
the CLP-treated rats showed a signicant increase in the lung
wet/dry weight ratio compared with the sham group. However,
the ratio was signicantly improved only in the group receiving
compound 26. The effect of compound 26 was further assayed
with regard to the intensity of the inammatory process. The
aim of the next phase of the study was to determine the total
protein content and level of leukocytes inltrated into the BALF
aer 24 h in the CLP group (Fig. 1C–E). The CLP-treated rats
showed increased levels of total protein and leukocytes
(mononuclear and polymorphonuclear) in the BALF compared
with the sham-treated group. The administration of compound
26 resulted in signicant inhibition of the total protein
concentration together with a decline in enhanced leukocytes
(mononuclear and polymorphonuclear) compared with the
CLP-treated group. In addition, the effect of compound 26
regarding MPO activity was also quantied, which is considered
to be an important biomarker for the activation of neutrophils
and their inltration. The results of this study are presented in
Fig. 1F. It was found that the CLP group had an enhanced level
of MPO activity, which was found to be signicantly reduced by
the administration of compound 26 at the maximum dose of
H
4-CH3
4-OCH3
4-Cl
4-F
H
4-CH3
4-OCH3
4-Cl
4-F
H
4-Cl
4-Cl
4-Cl
4-Cl
4-F
4-F
4-F
4-F
4-CH3
4-OCH3
4-Cl
4-F
H
4-CH3
4-OCH3
4-Cl
4-F
9.21
7.30
9.45
11.84
5.45
13.34
14.15
7.23
4-F
these results, it was suggested that uoro groups had a more
prominent inuence on the inhibitory prole, with less potency
in the case of chloro-containing compounds. These results
suggested that most of the compounds had better inhibitory
activity for MMP-8 than for MMP-2, with compound 26 being
the most potent analogue among the tested series.
20 mg kgꢀ1
.
Oxidative stress is a major determinant of ALI, which has
been reported to be associated with an inammatory response.
This releases the reactive oxygen free radicals and is deemed to
be toxic for cellular systems. Under normal circumstances, the
cellular system counteracts these free radicals by maintaining
In vivo pharmacological activity
Various studies have conrmed the role of MMP-8 in ALI,
against which our designed compounds showed excellent
inhibitory activity. Aer noting the exceptionally potent MMP-8
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Fig. 1 Effect of compound 26 in the CLP-induced septic rats. (A) Improvement in the lung histopathology in CLP-induced ALI in dose dependent
manner; (B) effect on lung wet/dry weight ratio; (C) total protein concentrations; (D) effect on the leukocytes of mononuclear and poly-
morphonuclear (E) counts in the BALF; (F) suppression of the MPO activity. Data were expressed as mean ꢁ standard deviation, where **p < 0.01,
significantly different from the sham group; #p < 0.05 and ##p < 0.01, significantly different from the CLP group.
Fig. 2 Effects of compound 26 on oxidative stress induced by CLP. Compound 26 causes significant reduction the MDA content (A) and
increases the SOD activity (B) together with GSH content (C) as suppressed by CLP. Data were expressed as mean ꢁ standard deviation, where
**p < 0.01, significantly different from the sham group; #p < 0.05 and ##p < 0.01, significantly different from the CLP group.
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Fig. 3 Effects of compound 26 on serum cytokine levels of CLP rats, as shown by decreased in the serum levels of TNF-a (a), IL-1b (b), and IL-6
(c) in the rats underwent CLP surgery. Data were expressed as mean ꢁ standard deviation, where **p < 0.01, significantly different from the sham
group; #p < 0.05 and ##p < 0.01, significantly different from the CLP group.
an optimal balance through the endogenous antioxidant the levels of TNF-a and IL-b were elevated in the serum of septic
system. The antioxidant defence system comprises MDA (a patients. Moreover, agents reducing the levels of these inam-
biomarker for oxidative stress-mediated lipid peroxidation), matory markers have a signicant effect on overall survival in
SOD (a superoxide scavenger), and GSH (a nonenzymatic anti- animal models. Thus, the anti-inammatory effect of
oxidant system). Thus, the effect of compound 26 on these compound 26 was assessed in terms of both systemic and
biomarkers was investigated to determine its effect on the pulmonary local inammatory responses, namely, TNF-a, IL-b,
antioxidant defence system. As shown in Fig. 2, the lungs of rats and IL-6. The results presented in Fig. 3 suggest that compound
in the CLP group had an enhanced level of MDA compared with 26 caused signicant reductions of the levels of TNF-a, IL-b, and
the level in the control group. Moreover, the levels of SOD and IL-6 and could protect the lung through modulation of systemic
GSH were found to be reduced in the CLP-treated lungs. The inammatory mediators in septic rats.
administration of compound 26 resulted in signicant attenu-
The levels of proinammatory enzymes, that is, COX-2 and
ation of these biomarkers; that is, the MDA level was found to be PGE2, are known to be elevated in ALI and other inammatory
reduced, while the levels of SOD and GSH were increased. Thus, diseases. The next stage of the study thus aimed at determining
it was suggested that compound 26 exerts a protective effect the effect of compound 26 on the expression of COX-2 and
against ALI via modulation of the antioxidant status.
PGE2. As shown in Fig. 4, compound 26 caused signicant dose-
Inammation plays a critical role in the progression of septic dependent inhibition of both COX-2 and PGE2 compared with
ALI, particularly when it occurs in the airways. It was found that their levels in the CLP-treated group.
Fig. 4 Effects of compound 26 on the inflammatory mediators in the lungs of CLP rats. The compound 26 causes notable down-regulation of
protein expression of COX-2 (b) and production of PGE2 (c) in the lungs of CLP rats as shown in representative western blot (a). Data were
expressed as mean ꢁ standard deviation, where **p < 0.01, significantly different from the sham group; #p < 0.05 and ##p < 0.01, significantly
different from the CLP group.
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Fig. 5 Effect of compound 26 on the NF-kB signaling pathway in the lung of CLP rats causes significant upregulation of protein expression of
IkBa (a), downregulates protein expression of NF-kB p65 (b) and p-p65 (c). Data were expressed as mean ꢁ standard deviation, where **p < 0.01,
significantly different from the sham group; #p < 0.05 and ##p < 0.01, significantly different from the CLP group.
Transcription of the genes encoding many inammatory conditions, NF-kB is stored in the cytoplasm by IkB proteins. In
proteins is mediated via NF-kB; thus, its inhibition offers the canonical pathway, IkB proteins are phosphorylated and
a
selective advantage in sepsis therapy. Under normal degraded by stimulus, resulting in the rapid translocation of
Fig. 6 Effect of compound 26 on the protein expression of Bcl-2 and Bax as determined by the western blot analysis. Data were expressed as mean ꢁ
standard deviation, where **p < 0.01, significantly different from the sham group; #p < 0.05 and ##p < 0.01, significantly different from the CLP group.
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Fig. 7 Effect of compound 26 on the expression of ICAM-1 and THP-1 cell adherence to activated A549 cells. The levels of ICAM-1 detected by
ELISA in LPS-activated A549 cells (a). Fluorescence microscopy images show THP-1 cells labelled with calcein-AM, mixed with normal or LPS-
activated A549 cells in absence or presence of compound 26 in different dose (b). Data were expressed as mean ꢁ standard deviation, where **p
< 0.01, significantly different from the sham group; #p < 0.05 and ##p < 0.01, significantly different from the CLP group.
active NF-kB complexes into the nucleus, where they initiate USA). Avance 400 and Avance 100 spectrometers (Billerica, MA,
target gene expression or repression. The results have been USA) were used for the determination of 1H NMR and 13C NMR
shown in Fig. 5. Compound 26 had a strong capacity to reduce spectral data, respectively, in ppm for chemical shis and hertz
the decrease of IkBa and increase NF-kB DNA binding activa- (Hz) for the coupling constant. The mass spectrum was ob-
tion. Thus, it was suggested that compound 26 had a protective tained using a liquid chromatography/mass spectrometry (LC/
effect on sepsis-induced ALI, possibly because of NF-kB inhi- MS) system (Waters ZQ; Waters, Milford, MA, USA) and an
bition. The effect of compound 26 on apoptotic markers was elemental analysis was conducted for C, H, and O using a Vario
also quantied, the results of which are presented in Fig. 6. It elemental analyser (Elementar, Langenselbold, Germany).
was found that compound 26 caused downregulation of the
expression levels of caspase-3 and Bax proteins. Intercellular
adhesion molecule-1 (ICAM-1) is considered to be a vital
General procedure for synthesis of 4-((4-substituted phenyl)
thiazole-2-yl)amine (3a–e)
adhesion molecule, inducing the migration of neutrophils and
A mixture of substituted acetophenone 1(a–e) (110 mmol) and
inltration during sepsis. In a recent study, it was concluded
thiourea (2) (220 mmol) in 100 mL of ethanol and then bromine
(110 mmol) was added in small fractions (0.5 mL) over 3 h, with
continuous stirring, and then heated on a steam bath for 8 h. A
sufficient quantity of water was then added and the solution was
that ICAM-1, results in the inhibition of sepsis-induced death
and lung injury. Thus, it was imperative to determine the effect
of compound 26 on ICAM-1, the results for which are presented
in Fig. 7a. It was found that compound 26 caused signicant
heated until all of the solid had dissolved. The solution was
inhibition of ICAM-1 expression in A549 cells compared with
ltered while hot. The ltrate was then cooled and concentrated
that in the lipopolysaccharide (LPS)-treated group. It was also
ammonia solution was added until precipitation ceased. The
found that the adherence of THP-1 cells to LPS-stimulated A549
solution was recrystallised with a pyridine–water (1 : 1) mixture
cells was inhibited, as shown in Fig. 7b.
to achieve a pure product 3(a–e).
4-Phenylthiazol-2-amine (3a). Yield: 86%; mp: 148–149 ꢂC;
MW: 176.24; Rf: 0.78; FT-IR (nmax; cmꢀ1 KBr): 3398 (N–H str,
Experimental
Chemicals
–NH), 3108 (Ar C–H str), 2842 (thiazole C–H str), 1621 (C]C str),
1516 (C]N str), 1139 (Ar–C–C str), 642 (C–S str) cmꢀ1; 1H-NMR
The starting materials and chemicals used in the processes of (400 MHz, DMSO, TMS) d ppm: 7.86 (d, 2H, J ¼ 7.9 Hz, Ar–H),
synthesis were obtained from Sigma Aldrich (St. Louis, MO, 7.38 (d, 2H, J ¼ 7.2 Hz, Ar–H), 7.31 (d, 1H, J ¼ 1.3 Hz, Ar–H), 7.15
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(s, 1H, thiazole-H), 6.94 (s, 2H, NH2); 13C-NMR (100 MHz, out and washed with distilled water, dried, and recrystallised
CDCl3) d, ppm: 168.7, 150.3, 133.1, 129.2, 128.8, 127.5, 101.8; from ethanol to give 4(a–e).
mass: 177.29 (M + 1); elemental analysis for C9H8N2S: calcu-
2-Chloro-N-(4-phenylthiazol-2-yl)acetamide (4a). Yield: 76%;
lated: C, 61.34; H, 4.58; N, 15.90; found: C, 61.39; H, 4.62; N, mp: 174–175 C; MW: 252.72; Rf: 0.79; FT-IR (nmax; cmꢀ1 KBr):
ꢂ
15.88.
3378 (N–H str, –NH), 3114 (Ar C–H str), 2882 (CH2), 2845
4-(p-tolyl)Thiazol-2-amine (3b). Yield: 88%; mp: 132–133 ꢂC; (thiazole C–H str), 1684 (C]O str), 1628 (C]C str), 1512 (C]N
MW: 190.26; Rf: 0.82; FT-IR (nmax; cmꢀ1 KBr): 3387 (N–H str, str), 1128 (Ar–C–C str), 762 (C–Cl str), 649 (C–S str) cmꢀ1; H-
1
–NH), 3104 (Ar C–H str), 2916 (C–H str, CH3), 2842 (thiazole C–H NMR (400 MHz, DMSO, TMS) d ppm: 9.16 (s, 1H, –CONH),
str), 1618 (C]C str), 1512 (C]N str), 1128 (Ar–C–C str), 647 (C–S 7.83 (d, 2H, J ¼ 7.9 Hz, Ar–H), 7.52 (s, 1H, thiazole-H), 7.46 (d,
str) cmꢀ1; 1H-NMR (400 MHz, DMSO, TMS) d ppm: 7.68 (d, 2H, J 2H, J ¼ 7.3 Hz, Ar–H), 7.38 (d, 1H, J ¼ 1.4 Hz, Ar–H), 4.42 (s, 2H,
¼ 8.2 Hz, Ar–H), 7.18 (d, 2H, J ¼ 8.1 Hz, Ar–H), 6.98 (s, 1H, CH2); 13C-NMR (100 MHz, CDCl3) d, ppm: 165.6, 164.3, 150.2,
thiazole-H), 6.94 (s, 2H, NH2), 2.29 (s, 3H, CH3); 13C-NMR (100 133.1, 129.3, 128.8, 127.5, 105.1, 42.8; mass: 253.78 (M + 1);
MHz, CDCl3) d, ppm: 168.9, 150.3, 131.8, 130.2, 129.6, 125.8, elemental analysis for C11H9ClN2OS: calculated: C, 52.28; H,
101.9, 21.4; mass: 191.24 (M + 1); elemental analysis for 3.59; N, 11.08; found: C, 52.31; H, 3.61; N, 11.04.
C
10H10N2S: calculated: C, 63.13; H, 5.30; N, 14.72; found: C,
2-Chloro-Nꢂ-(4-(p-tolyl)thiazol-2-yl)acetamide (4b). Yield: 72%;
63.18; H, 5.27; N, 14.78.
mp: 168–169 C; MW: 266.75; Rf: 0.68; FT-IR (nmax; cmꢀ1 KBr):
4-(4-Methoxyphenyl)thiazol-2-amine (3c). Yield: 81%; mp: 3373 (N–H str, –NH), 3116 (Ar C–H str), 2887 (CH2), 2848 (thiazole
204–205 C; MW: 206.26; Rf: 0.86; FT-IR (nmax; cmꢀ1 KBr): 3383 C–H str), 1687 (C]O str), 1624 (C]C str), 1516 (C]N str), 1126
(N–H str, –NH), 3106 (Ar C–H str), 2842 (thiazole C–H str), 1612 (Ar–C–C str), 765 (C–Cl str), 652 (C–S str) cmꢀ1 1H-NMR (400
(C]C str), 1518 (C]N str), 1129 (Ar–C–C str), 649 (C–S str) cmꢀ1
ꢂ
;
;
MHz, DMSO, TMS) d ppm: 9.15 (s, 1H, –CONH), 7.68 (d, 2H, J ¼
1H-NMR (400 MHz, DMSO, TMS) d ppm: 7.64 (d, 2H, J ¼ 8.8 Hz, 8.2 Hz, Ar–H), 7.54 (s, 1H, thiazole-H), 7.34 (d, 2H, J ¼ 7.6 Hz, Ar–
Ar–H), 7.12 (s, 1H, thiazole-H), 7.05 (d, 2H, J ¼ 8.4 Hz, Ar–H), 6.96 H), 4.41 (s, 2H, CH2), 2.35 (s, 3H, CH3); 13C-NMR (100 MHz,
(s, 2H, NH2), 3.84 (s, 3H, OCH3); 13C-NMR (100 MHz, CDCl3) CDCl3) d, ppm: 165.5, 164.3, 150.2, 131.8, 130.2, 129.6, 125.9,
d, ppm: 168.4, 160.6, 150.2, 128.5, 125.3, 114.8, 101.2, 55.8; mass: 105.2, 42.8, 21.4; mass: 267.72 (M + 1); elemental analysis for
207.29 (M + 1); elemental analysis for C10H10N2OS: calculated: C, C12H11ClN2OS: calculated: C, 54.03; H, 4.16; N, 10.50; found: C,
58.23; H, 4.89; N, 13.58; found: C, 58.28; H, 4.93; N, 13.63.
54.09; H, 4.23; N, 10.45.
4-(4-Chlorophenyl)thiazol-2-amine (3d). Yield: 78%; mp:
2-Chloro-N-(4-(4-methoxyphenyl)thiazol-2-yl)acetamide (4c).
168–169 ꢂC; MW: 210.68; Rf: 0.79; FT-IR (nmax; cmꢀ1 KBr): 3391 Yield: 77%; mp: 204–205 C; MW: 282.75; Rf: 0.64; FT-IR (nmax
;
ꢂ
(N–H str, –NH), 3108 (Ar C–H str), 2842 (thiazole C–H str), 1618 cmꢀ1 KBr): 3378 (N–H str, –NH), 3119 (Ar C–H str), 2882 (CH2),
(C]C str), 1512 (C]N str), 1125 (Ar–C–C str), 1094 (Ar–Cl 2841 (thiazole C–H str), 1692 (C]O str), 1627 (C]C str), 1512
1
stretch), 648 (C–S str) cmꢀ1; H-NMR (400 MHz, DMSO, TMS) (C]N str), 1128 (Ar–C–C str), 768 (C–Cl str), 657 (C–S str) cmꢀ1
;
d ppm: 7.69 (d, 2H, J ¼ 8.9 Hz, Ar–H), 7.58 (d, 2H, J ¼ 8.5 Hz, Ar– 1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.08 (s, 1H, –CONH),
H), 7.02 (s, 1H, thiazole-H), 6.95 (s, 2H, NH2); 13C-NMR (100 7.58 (d, 2H, J ¼ 8.8 Hz, Ar–H), 7.52 (s, 1H, thiazole-H), 7.18 (d,
MHz, CDCl3) d, ppm: 168.9, 150.4, 134.5, 131.2, 129.2, 128.8, 2H, J ¼ 7.2 Hz, Ar–H), 4.42 (s, 2H, CH2), 3.85 (s, 3H, CH3); 13C-
101.9; mass: 211.73 (M + 1); elemental analysis for C9H7ClN2S: NMR (100 MHz, CDCl3) d, ppm: 165.7, 164.3, 160.8, 150.4, 128.6,
calculated: C, 51.31; H, 3.35; N, 13.30; found: C, 51.26; H, 3.38; 125.4, 114.8, 105.2, 55.8, 42.9; mass: 283.82 (M + 1); elemental
N, 13.34.
analysis for C12H11ClN2O2S: calculated: C, 50.97; H, 3.92; N,
4-(4-Fluorophenyl)thiazol-2-amine (3e). Yield: 75%; mp: 115– 9.91; found: C, 50.94; H, 3.87; N, 9.98.
116 ꢂC; MW: 194.23; Rf: 0.73; FT-IR (nmax; cmꢀ1 KBr): 3385 (N–H
2-Chloro-N-(4-(4-chlorophenyl)thiazol-2-yl)acetamide (4d).
ꢂ
str, –NH), 3104 (Ar C–H str), 1612 (C]C str), 1521 (C]N str), Yield: 81%; mp: 195–196 C; MW: 287.17; Rf: 0.69; FT-IR (nmax
;
1118 (Ar–C–C str), 1156 (Ar–F), 645 (C–S str) cmꢀ1; 1H-NMR (400 cmꢀ1 KBr): 3372 (N–H str, –NH), 3115 (Ar C–H str), 2887 (CH2),
MHz, DMSO, TMS) d ppm: 8.07 (d, 2H, J ¼ 8.8 Hz, Ar–H), 7.09 (d, 2845 (thiazole C–H str), 1698 (C]O str), 1628 (C]C str), 1514
2H, J ¼ 8.2 Hz, Ar–H), 6.98 (s, 1H, thiazole-H), 6.94 (s, 2H, NH2); (C]N str), 1123 (Ar–C–C str), 762 (C–Cl str), 659 (C–S str) cmꢀ1
;
13C-NMR (100 MHz, CDCl3) d, ppm: 168.6, 162.8, 150.3, 130.8, 1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.12 (s, 1H, –CONH),
128.6, 116.2, 101.9; mass: 195.28 (M + 1); elemental analysis for 7.86 (d, 2H, J ¼ 8.7 Hz, Ar–H), 7.54 (s, 1H, thiazole-H), 7.48 (d,
C9H7FN2S: calculated: C, 55.65; H, 3.63; N, 14.42; found: C, 2H, J ¼ 7.1 Hz, Ar–H), 4.43 (s, 2H, CH2); 13C-NMR (100 MHz,
55.72; H, 3.68; N, 14.36.
CDCl3) d, ppm: 165.2, 164.1, 150.2, 134.4, 131.2, 129.3, 128.8,
105.2, 42.6; mass: 288.23 (M + 1); elemental analysis for C11
-
H8Cl2N2OS: calculated: C, 46.01; H, 2.81; N, 9.76; found: C,
46.04; H, 2.85; N, 9.72.
General procedure for synthesis of 2-chloro-N-[4-(p-
substituted phenyl)-thiazol-2-yl]-acetamides 4(a–e)
2-Chloro-N-(4-(4-uorophenyl)thiazol-2-yl)acetamide (4e).
ꢂ
Different substituted phenyl–thiazole derivatives 3(a–e) (110 Yield: 73%; mp: 212–213 C; MW: 270.71; Rf: 0.78; FT-IR (nmax
;
mmol) were dissolved in 20 mL of pyridine, with the dropwise cmꢀ1 KBr): 3378 (N–H str, –NH), 3118 (Ar C–H str), 2884 (CH2),
addition of chloroacetyl chloride (112 mmol) to the solution for 2842 (thiazole C–H str), 1697 (C]O str), 1627 (C]C str), 1516
30 min with continuous stirring. The mixture was then heated (C]N str), 1142 (Ar–F), 1128 (Ar–C–C str), 768 (C–Cl str), 654 (C–
on a water bath for 6 h. The mixture was cooled to room S str) cmꢀ1; 1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.14 (s, 1H,
temperature and then poured onto ice. The solid was separated –CONH), 8.15 (d, 2H, J ¼ 8.9 Hz, Ar–H), 7.51 (s, 1H, thiazole-H),
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7.34 (d, 2H, J ¼ 7.3 Hz, Ar–H), 4.42 (s, 2H, CH2); 13C-NMR (100 MHz, CDCl3) d, ppm: 168.6, 164.3, 152.9, 150.2, 146.2, 133.2,
MHz, CDCl3) d, ppm: 165.5, 164.3, 162.8, 150.4, 130.6, 128.6, 129.4, 128.8, 127.5, 115.3, 115.1, 105.2, 63.8, 55.8, 54.1, 51.8;
116.2, 105.2, 42.8; mass: 271.78 (M + 1); elemental analysis for mass: 409.54 (M + 1); elemental analysis for C22H24N4O2S:
C
11H8ClFN2OS: calculated: C, 48.80; H, 2.98; N, 10.35; found: C, calculated: C, 64.68; H, 5.92; N, 13.71; found: C, 64.72; H, 5.89;
48.84; H, 2.95; N, 10.42.
N, 13.78.
2-(4-(4-Chlorophenyl)piperazin-1-yl)-N-(4-phenylthiazol-2-yl)
acetamide (9). Yield: 77%; mp: 254–255 ꢂC; MW: 412.94; Rf:
0.69; FT-IR (nmax; cmꢀ1 KBr): 3392 (N–H str, –NH), 3128 (Ar C–H
General procedure for synthesis of title compounds 6–30
A mixture of different substituted 2-chloro-N-[4-(p-substituted str), 2881 (CH2), 2857 (thiazole C–H str), 1714 (C]O str), 1635
phenyl)-thiazol-2-yl]-acetamides 4(a–e) (10 mmol) was dissolved (C]C str), 1512 (C]N str), 1128 (Ar–C–C str), 769 (C–Cl str), 659
in 30 mL of ethanol and different substituted phenyl piperazine (C–S str) cmꢀ1; 1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.14 (s,
derivatives 5(i–v) (10 mmol) were added. The mixture was 1H, –CONH), 7.82 (d, 2H, J ¼ 7.8 Hz, Ar–H), 7.56 (s, 1H, thiazole-
reuxed for 9 h. Then, the mixture was cooled to room H), 7.36 (d, 2H, J ¼ 7.4 Hz, Ar–H), 7.28 (d, 1H, J ¼ 1.2 Hz, Ar–H),
temperature and poured into ice water. The product was 7.25 (d, 2H, J ¼ 8.2 Hz, Ar–H), 6.69 (d, 2H, J ¼ 7.1 Hz, Ar–H), 3.43
precipitated, washed with water, dried, and recrystallised from (dd, 4H, J ¼ 10.2 Hz, piperazine-H), 3.18 (s, 2H, CH2), 2.49 (dd,
ethanol to give title compounds 6–30.
4H, J ¼ 11.2 Hz, piperazine-H); 13C-NMR (100 MHz, CDCl3)
2-(4-Phenylpiperazin-1-yl)-N-(4-phenylthiazol-2-yl)acetamide d, ppm: 168.4, 164.3, 150.2, 147.9, 133.2, 129.8, 129.1, 128.6,
(6). Yield: 76%; mp: 218–219 ꢂC; MW: 378.49; Rf: 0.71; FT-IR 127.8, 127.2, 115.8, 105.2, 63.8, 54.2, 51.6; mass: 413.96 (M + 1);
(nmax; cmꢀ1 KBr): 3382 (N–H str, –NH), 3123 (Ar C–H str), 2886 elemental analysis for C21H21ClN4OS: calculated: C, 61.08; H,
(CH2), 2849 (thiazole C–H str), 1708 (C]O str), 1632 (C]C str), 5.13; N, 13.57; found: C, 61.04; H, 5.13; N, 13.56.
1519 (C]N str), 1124 (Ar–C–C str), 658 (C–S str) cmꢀ1; 1H-NMR
2-(4-(4-Fluorophenyl)piperazin-1-yl)-N-(4-phenylthiazol-2-yl)
(400 MHz, DMSO, TMS) d ppm: 9.16 (s, 1H, –CONH), 7.85 (d, 2H, acetamide (10). Yield: 71%; mp: 243–244 ꢂC; MW: 396.48; Rf:
J ¼ 7.9 Hz, Ar–H), 7.55 (s, 1H, thiazole-H), 7.34 (d, 2H, J ¼ 7.2 Hz, 0.62; FT-IR (nmax; cmꢀ1 KBr): 3391 (N–H str, –NH), 3124 (Ar C–H
Ar–H), 7.29 (d, 1H, J ¼ 1.4 Hz, Ar–H), 7.21 (d, 2H, J ¼ 8.2 Hz, Ar– str), 2887 (CH2), 2859 (thiazole C–H str), 1716 (C]O str), 1632
H), 6.84 (d, 2H, J ¼ 8.1 Hz, Ar–H), 6.72 (d, 1H, J ¼ 1.8 Hz, Ar–H), (C]C str), 1514 (C]N str), 1164 (Ar–F), 1123 (Ar–C–C str), 659
3.42 (dd, 4H, J ¼ 10.5 Hz, piperazine-H), 3.18 (s, 2H, CH2), 2.56 (C–S str) cmꢀ1; 1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.14 (s,
(dd, 4H, J ¼ 11.2 Hz, piperazine-H); 13C-NMR (100 MHz, CDCl3) 1H, –CONH), 7.84 (d, 2H, J ¼ 7.9 Hz, Ar–H), 7.55 (s, 1H, thiazole-
d, ppm: 168.7, 164.2, 150.4, 149.8, 133.2, 129.8, 129.2, 128.7, H), 7.39 (d, 2H, J ¼ 7.5 Hz, Ar–H), 7.29 (d, 1H, J ¼ 1.3 Hz, Ar–H),
127.5, 121.9, 114.3, 105.2, 63.8, 54.4, 51.6; mass: 379.56 (M + 1); 7.05 (d, 2H, J ¼ 8.1 Hz, Ar–H), 6.76 (d, 2H, J ¼ 7.4 Hz, Ar–H), 3.45
elemental analysis for C21H22N4OS: calculated: C, 66.64; H, 5.86; (dd, 4H, J ¼ 10.3 Hz, piperazine-H), 3.16 (s, 2H, CH2), 2.48 (dd,
N, 14.80; found: C, 66.68; H, 5.82; N, 14.78.
4H, J ¼ 11.2 Hz, piperazine-H); 13C-NMR (100 MHz, CDCl3)
N-(4-Phenylthiazol-2-yl)-2-(4-(p-tolyl)piperazin-1-yl)acetamide d, ppm: 168.4, 164.2, 156.8, 150.2, 145.2, 133.1, 129.4, 128.8,
(7). Yield: 83%; mp: 231–232 ꢂC; MW: 392.52; Rf: 0.67; FT-IR 127.6, 116.5, 115.8, 105.3, 63.6, 54.1, 51.8; mass: 397.51 (M + 1);
(nmax; cmꢀ1 KBr): 3392 (N–H str, –NH), 3129 (Ar C–H str), 2881 elemental analysis for C21H21FN4OS: calculated: C, 63.62; H,
(CH2), 2852 (thiazole C–H str), 1714 (C]O str), 1638 (C]C str), 5.34; N, 14.13; found: C, 63.61; H, 5.32; N, 14.15.
1513 (C]N str), 1126 (Ar–C–C str), 654 (C–S str) cmꢀ1; 1H-NMR
2-(4-Phenylpiperazin-1-yl)-N-(4-(p-tolyl)thiazol-2-yl)acetamide
ꢂ
(400 MHz, DMSO, TMS) d ppm: 9.14 (s, 1H, –CONH), 7.82 (d, 2H, (11). Yield: 83%; mp: 252–253 C; MW: 392.52; Rf: 0.69; FT-IR
J ¼ 7.8 Hz, Ar–H), 7.53 (s, 1H, thiazole-H), 7.37 (d, 2H, J ¼ 7.4 Hz, (nmax; cmꢀ1 KBr): 3394 (N–H str, –NH), 3129 (Ar C–H str), 2882
Ar–H), 7.28 (d, 1H, J ¼ 1.2 Hz, Ar–H), 7.08 (d, 2H, J ¼ 8.1 Hz, Ar– (CH2), 2851 (thiazole C–H str), 1718 (C]O str), 1634 (C]C str),
H), 6.58 (d, 2H, J ¼ 7.9 Hz, Ar–H), 3.45 (dd, 4H, J ¼ 10.2 Hz, 1516 (C]N str), 1128 (Ar–C–C str), 657 (C–S str) cmꢀ1; 1H-NMR
piperazine-H), 3.16 (s, 2H, CH2), 2.54 (dd, 4H, J ¼ 11.4 Hz, (400 MHz, DMSO, TMS) d ppm: 9.15 (s, 1H, –CONH), 7.82 (d, 2H,
piperazine-H), 2.28 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) J ¼ 7.8 Hz, Ar–H), 7.51 (s, 1H, thiazole-H), 7.34 (d, 2H, J ¼ 7.2 Hz,
d, ppm: 168.6, 164.3, 150.2, 146.8, 133.2, 130.8, 129.8, 129.2, Ar–H), 7.22 (d, 1H, J ¼ 7.1 Hz, Ar–H), 6.87 (d, 2H, J ¼ 8.2 Hz, Ar–
128.7, 127.5, 112.8, 105.1, 63.6, 54.2, 51.6, 21.3; mass: 393.58 (M H), 6.72 (d, 2H, J ¼ 1.2 Hz, Ar–H), 3.43 (dd, 4H, J ¼ 10.4 Hz,
+ 1); elemental analysis for C22H24N4OS: calculated: C, 67.32; H, piperazine-H), 3.19 (s, 2H, CH2), 2.46 (dd, 4H, J ¼ 11.3 Hz,
6.16; N, 14.27; found: C, 67.38; H, 6.12; N, 14.28.
piperazine-H), 2.28 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3)
2-(4-(4-Methoxyphenyl)piperazin-1-yl)-N-(4-phenylthiazol-2- d, ppm: 168.6, 164.4, 150.3, 149.7, 131.8, 130.2, 129.7, 129.4,
ꢂ
yl)acetamide (8). Yield: 73%; mp: 243–242 C; MW: 408.52; Rf: 125.8, 121.9, 114.4, 105.2, 63.8, 54.2, 51.8, 21.4; mass: 393.52 (M
0.63; FT-IR (nmax; cmꢀ1 KBr): 3394 (N–H str, –NH), 3123 (Ar C–H + 1); elemental analysis for C22H24N4OS: calculated: C, 67.32; H,
str), 2884 (CH2), 2858 (thiazole C–H str), 1718 (C]O str), 1632 6.16; N, 14.27; found: C, 67.31; H, 6.18; N, 14.26.
(C]C str), 1518 (C]N str), 1124 (Ar–C–C str), 657 (C–S str)
cmꢀ1
2-(4-(p-tolyl)Piperazin-1-yl)-N-(4-(p-tolyl)thiazol-2-yl)acetamide
;
1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.12 (s, 1H, (12). Yield: 69%; mp: 268–269 ꢂC; MW: 406.54; Rf: 0.84; FT-IR
–CONH), 7.83 (d, 2H, J ¼ 7.9 Hz, Ar–H), 7.54 (s, 1H, thiazole-H), (nmax; cmꢀ1 KBr): 3395 (N–H str, –NH), 3126 (Ar C–H str), 2884
7.34 (d, 2H, J ¼ 7.3 Hz, Ar–H), 7.29 (d, 1H, J ¼ 1.2 Hz, Ar–H), 6.87 (CH2), 2854 (thiazole C–H str), 1719 (C]O str), 1636 (C]C str),
1
(d, 2H, J ¼ 8.3 Hz, Ar–H), 6.63 (d, 2H, J ¼ 7.5 Hz, Ar–H), 3.94 (s, 1514 (C]N str), 1129 (Ar–C–C str), 659 (C–S str) cmꢀ1; H-NMR
3H, OCH3), 3.42 (dd, 4H, J ¼ 10.4 Hz, piperazine-H), 3.19 (s, 2H, (400 MHz, DMSO, TMS) d ppm: 9.14 (s, 1H, –CONH), 7.79 (d,
CH2), 2.52 (dd, 4H, J ¼ 11.2 Hz, piperazine-H); 13C-NMR (100 2H, J ¼ 7.9 Hz, Ar–H), 7.53 (s, 1H, thiazole-H), 7.29 (d, 2H, J ¼
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7.4 Hz, Ar–H), 7.04 (d, 2H, J ¼ 8.4 Hz, Ar–H), 6.68 (d, 2H, J ¼ str), 2889 (CH2), 2854 (thiazole C–H str), 1716 (C]O str), 1638
1.4 Hz, Ar–H), 3.45 (dd, 4H, J ¼ 10.3 Hz, piperazine-H), 3.18 (s, (C]C str), 1517 (C]N str), 1126 (Ar–C–C str), 654 (C–S str)
2H, CH2), 2.48 (dd, 4H, J ¼ 11.2 Hz, piperazine-H), 2.31 (s, 3H, cmꢀ1
;
1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.14 (s, 1H,
CH3), 2.29 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) d, ppm: –CONH), 7.56 (d, 2H, J ¼ 8.8 Hz, Ar–H), 7.52 (s, 1H, thiazole-H),
168.6, 164.3, 150.2, 146.8, 131.8, 130.9, 130.2, 129.8, 129.3, 125.7, 7.27 (d, 2H, J ¼ 7.6 Hz, Ar–H), 7.02 (d, 2H, J ¼ 8.2 Hz, Ar–H), 6.87
112.8, 105.1, 63.8, 54.1, 51.8, 21.3; mass: 407.55 (M + 1); (d, 2H, J ¼ 8.1 Hz, Ar–H), 6.74 (d, 1H, J ¼ 1.4 Hz, Ar–H), 3.87 (s,
elemental analysis for C23H26N4OS: calculated: C, 67.95; H, 6.45; 3H, OCH3), 3.45 (dd, 4H, J ¼ 10.4 Hz, piperazine-H), 3.19 (s, 2H,
N, 13.78; found: C, 67.97; H, 6.44; N, 13.79.
CH2), 2.49 (dd, 4H, J ¼ 11.3 Hz, piperazine-H); 13C-NMR (100
2-(4-(4-Methoxyphenyl)piperazin-1-yl)-N-(4-(p-tolyl)thiazol-2- MHz, CDCl3) d, ppm: 168.6, 164.3, 160.8, 150.4, 149.8, 129.8,
yl)acetamide (13). Yield: 75%; mp: 289–290 ꢂC; MW: 422.54; Rf: 128.4, 125.4, 121.9, 114.9, 114.2, 105.2, 63.8, 55.8, 54.2, 51.8;
0.80; FT-IR (nmax; cmꢀ1 KBr): 3398 (N–H str, –NH), 3122 (Ar C–H mass: 409.53 (M + 1); elemental analysis for C22H24N4O2S:
str), 2882 (CH2), 2851 (thiazole C–H str), 1716 (C]O str), 1638 calculated: C, 64.68; H, 5.92; N, 13.71; found: C, 64.70; H, 5.91;
(C]C str), 1512 (C]N str), 1124 (Ar–C–C str), 658 (C–S str) N, 13.72.
cmꢀ1
;
1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.15 (s, 1H,
N-(4-(4-Methoxyphenyl)thiazol-2-yl)-2-(4-(p-tolyl)piperazin-1-
–CONH), 7.81 (d, 2H, J ¼ 7.8 Hz, Ar–H), 7.56 (s, 1H, thiazole-H), yl)acetamide (17). Yield: 76%; mp: 297–298 ꢂC; MW: 422.54; Rf:
7.31 (d, 2H, J ¼ 7.3 Hz, Ar–H), 6.92 (d, 2H, J ¼ 8.2 Hz, Ar–H), 6.65 0.71; FT-IR (nmax; cmꢀ1 KBr): 3398 (N–H str, –NH), 3123 (Ar C–H
(d, 2H, J ¼ 1.3 Hz, Ar–H), 3.87 (s, 3H, OCH3) 3.43 (dd, 4H, J ¼ str), 2887 (CH2), 2859 (thiazole C–H str), 1718 (C]O str), 1639
10.2 Hz, piperazine-H), 3.19 (s, 2H, CH2), 2.46 (dd, 4H, J ¼ (C]C str), 1516 (C]N str), 1129 (Ar–C–C str), 657 (C–S str)
11.2 Hz, piperazine-H), 2.34 (s, 3H, CH3); 13C-NMR (100 MHz, cmꢀ1 1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.15 (s, 1H,
;
CDCl3) d, ppm: 168.6, 164.2, 152.8, 150.3, 146.4, 131.8, 130.1, –CONH), 7.55 (d, 2H, J ¼ 8.7 Hz, Ar–H), 7.54 (s, 1H, thiazole-H),
129.6, 125.8, 115.4, 115.1, 105.1, 63.8, 54.2, 51.8, 56.8, 21.4; 7.06 (d, 2H, J ¼ 8.2 Hz, Ar–H), 7.03 (d, 2H, J ¼ 8.1 Hz, Ar–H), 6.69
mass: 423.56 (M + 1); elemental analysis for C23H26N4O2S: (d, 2H, J ¼ 8.4 Hz, Ar–H), 3.85 (s, 3H, OCH3), 3.46 (dd, 4H, J ¼
calculated: C, 65.38; H, 6.20; N, 13.26; found: C, 65.36; H, 6.20; 10.3 Hz, piperazine-H), 3.18 (s, 2H, CH2), 2.47 (dd, 4H, J ¼
N, 13.28.
11.2 Hz, piperazine-H), 2.35 (s, 3H, CH3); 13C-NMR (100 MHz,
2-(4-(4-Chlorophenyl)piperazin-1-yl)-N-(4-(p-tolyl)thiazol-2- CDCl3) d, ppm: 168.7, 164.4, 160.7, 150.3, 146.6, 130.8, 129.9,
yl)acetamide (14). Yield: 79%; mp: 282–283 ꢂC; MW: 426.96; Rf: 128.6, 125.4, 114.9, 112.8, 105.2, 63.8, 55.8, 54.1, 51.6, 21.3;
0.71; FT-IR (nmax; cmꢀ1 KBr): 3394 (N–H str, –NH), 3128 (Ar C–H mass: 423.55 (M + 1); elemental analysis for C23H26N4O2S:
str), 2881 (CH2), 2855 (thiazole C–H str), 1719 (C]O str), 1632 calculated: C, 65.38; H, 6.20; N, 13.26; found: C, 65.40; H, 6.18;
(C]C str), 1518 (C]N str), 1126 (Ar–C–C str), 773 (C–Cl str), 656 N, 13.27.
(C–S str) cmꢀ1; 1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.14 (s,
2-(4-(4-Methoxyphenyl)piperazin-1-yl)-N-(4-(4-methoxyphenyl)-
1H, –CONH), 7.78 (d, 2H, J ¼ 7.9 Hz, Ar–H), 7.54 (s, 1H, thiazole- thiazol-2-yl)acetamide (18). Yield: 82%; mp: 302–303 ꢂC; MW:
H), 7.30 (d, 2H, J ¼ 7.4 Hz, Ar–H), 7.24 (d, 2H, J ¼ 8.4 Hz, Ar–H), 438.54; Rf: 0.79; FT-IR (nmax; cmꢀ1 KBr): 3392 (N–H str, –NH), 3125
6.69 (d, 2H, J ¼ 1.4 Hz, Ar–H), 3.45 (dd, 4H, J ¼ 10.3 Hz, (Ar C–H str), 2889 (CH2), 2852 (thiazole C–H str), 1714 (C]O str),
piperazine-H), 3.18 (s, 2H, CH2), 2.48 (dd, 4H, J ¼ 11.3 Hz, 1637 (C]C str), 1518 (C]N str), 1127 (Ar–C–C str), 659 (C–S str)
piperazine-H), 2.32 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) cmꢀ1 1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.14 (s, 1H,
;
d, ppm: 168.6, 164.3, 150.2, 147.8, 131.8, 130.2, 129.8, 129.4, –CONH), 7.58 (d, 2H, J ¼ 8.8 Hz, Ar–H), 7.55 (s, 1H, thiazole-H),
127.2, 125.8, 115.7, 105.1, 63.8, 54.1, 51.7, 21.4; mass: 427.98 (M 7.03 (d, 2H, J ¼ 8.1 Hz, Ar–H), 6.87 (d, 2H, J ¼ 8.6 Hz, Ar–H),
+ 1); elemental analysis for C22H23ClN4OS: calculated: C, 61.89; 6.64 (d, 2H, J ¼ 8.3 Hz, Ar–H), 3.85 (s, 3H, OCH3), 3.78 (s, 3H,
H, 5.43; N, 13.12; found: C, 61.91; H, 5.42; N, 13.12.
OCH3), 3.45 (dd, 4H, J ¼ 10.2 Hz, piperazine-H), 3.19 (s, 2H, CH2),
2-(4-(4-Fluorophenyl)piperazin-1-yl)-N-(4-(p-tolyl)thiazol-2- 2.48 (dd, 4H, J ¼ 11.2 Hz, piperazine-H); 13C-NMR (100 MHz,
yl)acetamide (15). Yield: 72%; mp: 269–270 ꢂC; MW: 410.51; Rf: CDCl3) d, ppm: 168.6, 164.3, 160.8, 152.9, 150.3, 146.2, 128.7,
0.73; FT-IR (nmax; cmꢀ1 KBr): 3391 (N–H str, –NH), 3124 (Ar C–H 125.4, 115.3, 115.2, 114.9, 105.2, 63.8, 55.8, 54.2, 51.6; mass:
str), 2882 (CH2), 2858 (thiazole C–H str), 1714 (C]O str), 1636 439.54 (M + 1); elemental analysis for C23H26N4O3S: calculated: C,
(C]C str), 1519 (C]N str), 1148 (Ar–F), 1128 (Ar–C–C str), 658 62.99; H, 5.98; N, 12.78; found: C, 62.96; H, 5.98; N, 12.77.
(C–S str) cmꢀ1; 1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.14 (s,
2-(4-(4-Chlorophenyl)piperazin-1-yl)-N-(4-(4-methoxyphenyl)
1H, –CONH), 7.76 (d, 2H, J ¼ 7.8 Hz, Ar–H), 7.55 (s, 1H, thiazole- thiazol-2-yl)acetamide (19). Yield: 75%; mp: 293–294 ꢂC; MW:
H), 7.31 (d, 2H, J ¼ 7.3 Hz, Ar–H), 7.04 (d, 2H, J ¼ 8.1 Hz, Ar–H), 442.96; Rf: 0.83; FT-IR (nmax; cmꢀ1 KBr): 3389 (N–H str, –NH),
6.84 (d, 2H, J ¼ 1.2 Hz, Ar–H), 3.43 (dd, 4H, J ¼ 10.2 Hz, 3122 (Ar C–H str), 2886 (CH2), 2857 (thiazole C–H str), 1718 (C]
piperazine-H), 3.16 (s, 2H, CH2), 2.46 (dd, 4H, J ¼ 11.2 Hz, O str), 1630 (C]C str), 1512 (C]N str), 1129 (Ar–C–C str), 783
piperazine-H), 2.31 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) (C–Cl str), 657 (C–S str) cmꢀ1; 1H-NMR (400 MHz, DMSO, TMS)
d, ppm: 168.7, 164.3, 156.8, 150.4, 145.2, 131.9, 130.2, 129.6, d ppm: 9.14 (s, 1H, –CONH), 7.56 (d, 2H, J ¼ 8.9 Hz, Ar–H), 7.54
125.9, 116.4, 115.9, 105.2, 63.8, 54.1, 51.8, 21.4; mass: 411.52 (M (s, 1H, thiazole-H), 7.28 (d, 2H, J ¼ 8.2 Hz, Ar–H), 7.04 (d, 2H, J ¼
+ 1); elemental analysis for C22H23FN4OS: calculated: C, 64.37; 8.1 Hz, Ar–H), 6.71 (d, 2H, J ¼ 8.5 Hz, Ar–H), 3.83 (s, 3H, OCH3),
H, 5.65; N, 13.65; found: C, 64.39; H, 5.65; N, 13.64.
3.43 (dd, 4H, J ¼ 10.4 Hz, piperazine-H), 3.18 (s, 2H, CH2), 2.47
N-(4-(4-Methoxyphenyl)thiazol-2-yl)-2-(4-phenylpiperazin-1- (dd, 4H, J ¼ 11.1 Hz, piperazine-H); 13C-NMR (100 MHz, CDCl3)
yl)acetamide (16). Yield: 86%; mp: 281–282 ꢂC; MW: 408.52; Rf: d, ppm: 168.7, 164.3, 160.8, 150.2, 147.7, 129.8, 128.4, 127.2,
0.79; FT-IR (nmax; cmꢀ1 KBr): 3394 (N–H str, –NH), 3128 (Ar C–H 125.4, 115.7, 114.8, 105.1, 63.7, 54.1, 51.6, 55.8; mass: 443.97
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(M + 1); elemental analysis for C22H23ClN4O2S: calculated: C, (dd, 4H, J ¼ 11.3 Hz, piperazine-H); 13C-NMR (100 MHz, CDCl3)
59.65; H, 5.23; N, 12.65; found: C, 59.67; H, 5.23; N, 12.66.
d, ppm: 168.6, 164.3, 152.9, 150.2, 146.4, 134.4, 131.2, 129.4,
2-(4-(4-Fluorophenyl)piperazin-1-yl)-N-(4-(4-methoxyphenyl) 128.8, 115.4, 115.1, 105.2, 63.7, 55.8, 54.2, 51.8; mass: 443.97 (M
thiazol-2-yl)acetamide (20). Yield: 77%; mp: 309–310 ꢂC; MW: + 1); elemental analysis for C22H23ClN4O2S: calculated: C, 59.65;
426.51; Rf: 0.86; FT-IR (nmax; cmꢀ1 KBr): 3381 (N–H str, –NH), H, 5.23; N, 12.65; found: C, 59.67; H, 5.22; N, 12.67.
3128 (Ar C–H str), 2884 (CH2), 2859 (thiazole C–H str), 1712 (C]
O str), 1631 (C]C str), 1516 (C]N str), 1168 (Ar–F), 1121 (Ar–C– thiazol-2-yl)acetamide (24). Yield: 79%; mp: 313–314 ꢂC; MW:
C str), 658 (C–S str) cmꢀ1 1H-NMR (400 MHz, DMSO, TMS) 447.38; Rf: 0.85; FT-IR (nmax; cmꢀ1 KBr): 3398 (N–H str, –NH),
2-(4-(4-Chlorophenyl)piperazin-1-yl)-N-(4-(4-chlorophenyl)
;
d ppm: 9.15 (s, 1H, –CONH), 7.54 (d, 2H, J ¼ 8.7 Hz, Ar–H), 7.56 3137 (Ar C–H str), 2885 (CH2), 2857 (thiazole C–H str), 1718 (C]
(s, 1H, thiazole-H), 7.09 (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.03 (d, 2H, J ¼ O str), 1634 (C]C str), 1514 (C]N str), 1128 (Ar–C–C str), 792
8.2 Hz, Ar–H), 6.75 (d, 2H, J ¼ 8.4 Hz, Ar–H), 3.85 (s, 3H, OCH3), (C–Cl str), 659 (C–S str) cmꢀ1; 1H-NMR (400 MHz, DMSO, TMS)
3.45 (dd, 4H, J ¼ 10.2 Hz, piperazine-H), 3.17 (s, 2H, CH2), 2.48 d ppm: 9.15 (s, 1H, –CONH), 8.03 (d, 2H, J ¼ 8.8 Hz, Ar–H), 7.55
(dd, 4H, J ¼ 11.2 Hz, piperazine-H); 13C-NMR (100 MHz, CDCl3) (s, 1H, thiazole-H), 7.46 (d, 2H, J ¼ 8.2 Hz, Ar–H), 7.28 (d, 2H, J ¼
d, ppm: 168.6, 164.3, 160.7, 156.8, 150.2, 145.3, 128.6, 125.4, 8.5 Hz, Ar–H), 6.62 (d, 2H, J ¼ 7.8 Hz, Ar–H), 3.43 (dd, 4H, J ¼
116.5, 115.8, 114.9, 105.2, 63.7, 55.8, 54.2, 51.8; mass: 427.52 (M 10.2 Hz, piperazine-H), 3.18 (s, 2H, CH2), 2.49 (dd, 4H, J ¼
+ 1); elemental analysis for C22H23FN4O2S: calculated: C, 61.95; 11.2 Hz, piperazine-H); 13C-NMR (100 MHz, CDCl3) d, ppm:
H, 5.44; N, 13.14; found: C, 61.97; H, 5.42; N, 13.14.
168.6, 164.3, 150.3, 147.7, 134.4, 131.2, 129.8, 129.2, 128.9,
N-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(4-phenylpiperazin-1-yl) 127.2, 115.7, 105.1, 63.7, 54.2, 51.8; mass: 448.37 (M + 1);
acetamide (21). Yield: 71%; mp: 289–290 ꢂC; MW: 412.94; Rf: elemental analysis for C21H20Cl2N4OS: calculated: C, 56.38; H,
0.81; FT-IR (nmax; cmꢀ1 KBr): 3387 (N–H str, –NH), 3129 (Ar C–H 4.51; N, 12.52; found: C, 56.37; H, 4.52; N, 12.51.
str), 2883 (CH2), 2857 (thiazole C–H str), 1718 (C]O str), 1635
N-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(4-(4-uorophenyl)pip-
(C]C str), 1517 (C]N str), 1125 (Ar–C–C str), 792 (C–Cl str), 652 erazin-1-yl)acetamide (25). Yield: 83%; mp: 318–319 ꢂC; MW:
(C–S str) cmꢀ1; 1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.14 (s, 430.93; Rf: 0.81; FT-IR (nmax; cmꢀ1 KBr): 3392 (N–H str, –NH),
1H, –CONH), 8.02 (d, 2H, J ¼ 8.9 Hz, Ar–H), 7.54 (s, 1H, thiazole- 3139 (Ar C–H str), 2882 (CH2), 2859 (thiazole C–H str), 1714 (C]
H), 7.42 (d, 2H, J ¼ 7.8 Hz, Ar–H), 7.23 (d, 2H, J ¼ 8.8 Hz, Ar–H), O str), 1632 (C]C str), 1518 (C]N str), 1174 (Ar–F), 1121 (Ar–C–
1
6.87 (d, 2H, J ¼ 8.2 Hz, Ar–H), 6.68 (d, 1H, J ¼ 1.3 Hz, Ar–H), 3.43 C str), 798 (C–Cl str), 657 (C–S str) cmꢀ1; H-NMR (400 MHz,
(dd, 4H, J ¼ 10.3 Hz, piperazine-H), 3.19 (s, 2H, CH2), 2.46 (dd, DMSO, TMS) d ppm: 9.14 (s, 1H, –CONH), 8.02 (d, 2H, J ¼
4H, J ¼ 11.1 Hz, piperazine-H); 13C-NMR (100 MHz, CDCl3) 8.9 Hz, Ar–H), 7.53 (s, 1H, thiazole-H), 7.48 (d, 2H, J ¼ 8.4 Hz,
d, ppm: 168.7, 164.3, 150.4, 149.7, 134.4, 131.2, 129.7, 129.4, Ar–H), 7.04 (d, 2H, J ¼ 8.8 Hz, Ar–H), 6.65 (d, 2H, J ¼ 7.6 Hz, Ar–
128.8, 121.9, 114.4, 105.2, 63.7, 54.1, 51.7; mass: 413.96 (M + 1); H), 3.45 (dd, 4H, J ¼ 10.3 Hz, piperazine-H), 3.17 (s, 2H, CH2),
elemental analysis for C21H21ClN4OS: calculated: C, 61.08; H, 2.47 (dd, 4H, J ¼ 11.3 Hz, piperazine-H); 13C-NMR (100 MHz,
5.13; N, 13.57; found: C, 61.06; H, 5.13; N, 13.57.
CDCl3) d, ppm: 168.4, 164.3, 156.9, 150.3, 145.3, 134.4, 131.2,
N-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(4-(p-tolyl)piperazin-1- 129.4, 128.9, 116.5, 115.8, 105.3, 63.7, 54.2, 51.8; mass: 431.95
yl)acetamide (22). Yield: 82%; mp: 305–306 ꢂC; MW: 426.96; Rf: (M + 1); elemental analysis for C21H20ClFN4OS: calculated: C,
0.89; FT-IR (nmax; cmꢀ1 KBr): 3393 (N–H str, –NH), 3135 (Ar C–H 58.53; H, 4.68; N, 13.00; found: C, 58.55; H, 4.69; N, 13.01.
str), 2889 (CH2), 2852 (thiazole C–H str), 1713 (C]O str), 1632
N-(4-(4-Fluorophenyl)thiazol-2-yl)-2-(4-phenylpiperazin-1-yl)
(C]C str), 1519 (C]N str), 1121 (Ar–C–C str), 798 (C–Cl str), 654 acetamide (26). Yield: 80%; mp: 281–282 ꢂC; MW: 396.48; Rf:
(C–S str) cmꢀ1; 1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.16 (s, 0.56; FT-IR (nmax; cmꢀ1 KBr): 3398 (N–H str, –NH), 3142 (Ar C–H
1H, –CONH), 8.03 (d, 2H, J ¼ 8.8 Hz, Ar–H), 7.56 (s, 1H, thiazole- str), 2888 (CH2), 2854 (thiazole C–H str), 1718 (C]O str), 1639
H), 7.46 (d, 2H, J ¼ 7.9 Hz, Ar–H), 7.04 (d, 2H, J ¼ 8.2 Hz, Ar–H), (C]C str), 1512 (C]N str), 1187 (Ar–F), 654 (C–S str) cmꢀ1; 1H-
6.62 (d, 2H, J ¼ 8.1 Hz, Ar–H), 3.45 (dd, 4H, J ¼ 10.4 Hz, NMR (400 MHz, DMSO, TMS) d ppm: 9.13 (s, 1H, –CONH), 8.14
piperazine-H), 3.18 (s, 2H, CH2), 2.45 (dd, 4H, J ¼ 11.2 Hz, (d, 2H, J ¼ 8.8 Hz, Ar–H), 7.56 (s, 1H, thiazole-H), 7.28 (d, 2H, J ¼
piperazine-H), 2.28 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) 8.3 Hz, Ar–H), 7.18 (d, 2H, J ¼ 8.2 Hz, Ar–H), 6.96 (d, 2H, J ¼
d, ppm: 168.7, 164.3, 150.3, 146.7, 134.4, 131.2, 130.8, 129.8, 8.1 Hz, Ar–H), 6.72 (d, 1H, J ¼ 1.4 Hz, Ar–H), 3.46 (dd, 4H, J ¼
129.3, 128.7, 112.9, 105.3, 63.7, 54.3, 51.7, 21.4; mass: 427.98 (M 10.2 Hz, piperazine-H), 3.18 (s, 2H, CH2), 2.44 (dd, 4H, J ¼
+ 1); elemental analysis for C22H23ClN4OS: calculated: C, 61.89; 11.2 Hz, piperazine-H); 13C-NMR (100 MHz, CDCl3) d, ppm:
H, 5.43; N, 13.12; found: C, 61.91; H, 5.45; N, 13.12.
168.7, 164.3, 162.8, 150.2, 149.8, 130.7, 129.8, 128.6, 121.9,
N-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(4-(4-methoxyphenyl) 116.2, 114.3, 105.2, 63.7, 54.2, 51.6; mass: 397.49 (M + 1);
piperazin-1-yl)acetamide (23). Yield: 73%; mp: 323–324 ꢂC; MW: elemental analysis for C21H21FN4OS: calculated: C, 63.62; H,
442.96; Rf: 0.81; FT-IR (nmax; cmꢀ1 KBr): 3395 (N–H str, –NH), 5.34; N, 14.13; found: C, 63.63; H, 5.34; N, 14.11.
3132 (Ar C–H str), 2881 (CH2), 2855 (thiazole C–H str), 1713 (C]
N-(4-(4-Fluorophenyl)thiazol-2-yl)-2-(4-(p-tolyl)piperazin-1-yl)-
O str), 1631 (C]C str), 1517 (C]N str), 1125 (Ar–C–C str), 794 acetamide (27). Yield: 87%; mp: 309–310 ꢂC; MW: 410.51; Rf:
(C–Cl str), 657 (C–S str) cmꢀ1; 1H-NMR (400 MHz, DMSO, TMS) 0.59; FT-IR (nmax; cmꢀ1 KBr): 3392 (N–H str, –NH), 3149 (Ar C–H
d ppm: 9.14 (s, 1H, –CONH), 8.02 (d, 2H, J ¼ 8.9 Hz, Ar–H), 7.57 str), 2881 (CH2), 2857 (thiazole C–H str), 1714 (C]O str), 1643
(s, 1H, thiazole-H), 7.48 (d, 2H, J ¼ 8.1 Hz, Ar–H), 6.84 (d, 2H, J ¼ (C]C str), 1514 (C]N str), 1189 (Ar–F), 658 (C–S str) cmꢀ1; 1H-
8.3 Hz, Ar–H), 6.58 (d, 2H, J ¼ 8.1 Hz, Ar–H), 3.85 (s, 3H, OCH3), NMR (400 MHz, DMSO, TMS) d ppm: 9.15 (s, 1H, –CONH), 8.12
3.44 (dd, 4H, J ¼ 10.3 Hz, piperazine-H), 3.17 (s, 2H, CH2), 2.47 (d, 2H, J ¼ 8.9 Hz, Ar–H), 7.57 (s, 1H, thiazole-H), 7.29 (d, 2H, J ¼
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8.2 Hz, Ar–H), 7.03 (d, 2H, J ¼ 8.1 Hz, Ar–H), 6.58 (d, 2H, J ¼ 902-MP and 908-MP, respectively, and with uorogenic peptide
7.8 Hz, Ar–H), 3.45 (dd, 4H, J ¼ 10.4 Hz, piperazine-H), 3.17 (s, QF24 as a substrate for the in situ determination of MMP. The
2H, CH2), 2.42 (dd, 4H, J ¼ 11.2 Hz, piperazine-H), 2.28 (s, 3H, proenzymes (pro-MMP-2 and -8) were activated immediately
CH3); 13C-NMR (100 MHz, CDCl3) d, ppm: 168.4, 164.3, 162.8, before their use with p-aminophenylmercuric acetate (APMA, 1
ꢀ2
ꢂ
150.2, 146.7, 130.9, 130.5, 129.9, 128.7, 116.2, 112.9, 105.2, 63.7, mM) for 1 h at 37 C. Briey, 10 mM solutions of the inhib-
54.1, 51.6, 21.5; mass: 411.52 (M + 1); elemental analysis for itors were made in 5% MeOH/DMSO and further diluted as
C
22H23FN4OS: calculated: C, 64.37; H, 5.65; N, 13.65; found: C, required in the assay buffer (50 mM Tris–HCl, 5 mM CaCl2,
64.39; H, 5.67; N, 13.65. 0.02% NaN3, 0.05% Brij 35, pH 7.4, for MMP-2; and 50 mM Tris–
N-(4-(4-Fluorophenyl)thiazol-2-yl)-2-(4-(4-methoxyphenyl) HCl, 10 mM CaCl2, 150 mM NaCl, pH 7.5, for MMP-8). The
piperazin-1-yl)acetamide (28). Yield: 84%; mp: 314–315 ꢂC; MW: activated enzyme together with inhibitor solution was incu-
426.51; Rf: 0.53; FT-IR (nmax; cmꢀ1 KBr): 3396 (N–H str, –NH), bated in the assay buffer for 3 h, and the temperature was
ꢂ
ꢂ
3144 (Ar C–H str), 2883 (CH2), 2856 (thiazole C–H str), 1715 (C] maintained at 25 C for MMP-2 and at 37 C for MMP-8. Aer
O str), 1648 (C]C str), 1512 (C]N str), 1193 (Ar–F), 659 (C–S str) the addition of a 0.054 mM DMSO solution of uorogenic
cmꢀ1 1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.13 (s, 1H, substrate QF24, hydrolysis was monitored by recording the
;
–CONH), 8.16 (d, 2H, J ¼ 8.8 Hz, Ar–H), 7.55 (s, 1H, thiazole-H), increase in uorescence (lex 328 and 393 nm) during a 30 min
7.28 (d, 2H, J ¼ 8.3 Hz, Ar–H), 6.83 (d, 2H, J ¼ 8.2 Hz, Ar–H), 6.54 period using a spectrouorometer. The IC50 values were calcu-
(d, 2H, J ¼ 7.9 Hz, Ar–H), 3.84 (s, 3H, OCH3), 3.46 (dd, 4H, J ¼ lated from control reactions without the inhibitor and per-
10.2 Hz, piperazine-H), 3.19 (s, 2H, CH2), 2.45 (dd, 4H, J ¼ formed in triplicate.
11.1 Hz, piperazine-H); 13C-NMR (100 MHz, CDCl3) d, ppm:
168.6, 164.3, 162.8, 152.8, 150.3, 146.4, 130.8, 128.7, 116.2,
115.6, 115.3, 105.2, 63.7, 56.9, 54.2, 51.8; mass: 427.53 (M + 1);
Pharmacological evaluation
elemental analysis for C22H23FN4O2S: calculated: C, 61.95; H,
5.44; N, 13.14; found: C, 61.97; H, 5.43; N, 13.15.
Animals. Male Sprague-Dawley rats (6–8 weeks old, weighing
200–250 g) were obtained from the animal house of Soochow
2-(4-(4-Chlorophenyl)piperazin-1-yl)-N-(4-(4-uorophenyl) University Affiliated Children's Hospital, China. All experiments
thiazol-2-yl)acetamide (29). Yield: 88%; mp: 325–326 ꢂC; MW: were performed in accordance with the guidelines for the use of
430.93; Rf: 0.56; FT-IR (nmax; cmꢀ1 KBr): 3393 (N–H str, –NH), experimental animals by the National Institutes of Health
3142 (Ar C–H str), 2881 (CH2), 2858 (thiazole C–H str), 1719 (C] (Bethesda, MD) and were approved by the Institutional Animal
O str), 1649 (C]C str), 1517 (C]N str), 1195 (Ar–F), 796 (C–Cl Care and Use Committee of Soochow University Affiliated
str), 653 (C–S str) cmꢀ1; 1H-NMR (400 MHz, DMSO, TMS) d ppm: Children's Hospital, China. Rats were kept at a temperature of
9.14 (s, 1H, –CONH), 8.14 (d, 2H, J ¼ 8.9 Hz, Ar–H), 7.56 (s, 1H, 22 ꢂC, relative humidity of 50–60%, and alternate light–dark
thiazole-H), 7.29 (d, 2H, J ¼ 8.4 Hz, Ar–H), 7.24 (d, 2H, J ¼ (12/12 h) conditions. The animals were provided standard
8.1 Hz, Ar–H), 6.63 (d, 2H, J ¼ 7.8 Hz, Ar–H), 3.48 (dd, 4H, J ¼ laboratory food and water ad libitum.
10.3 Hz, piperazine-H), 3.16 (s, 2H, CH2), 2.43 (dd, 4H, J ¼
Establishment of sepsis model. All rats were randomly
11.2 Hz, piperazine-H); 13C-NMR (100 MHz, CDCl3) d, ppm: divided into four groups:
168.4, 164.3, 162.8, 150.2, 147.8, 130.8, 129.8, 128.7, 127.3,
116.2, 115.8, 105.2, 63.7, 54.1, 51.6; mass: 431.93 (M + 1);
elemental analysis for C21H20ClFN4OS: calculated: C, 58.53; H,
4.68; N, 13.00; found: C, 58.55; H, 4.70; N, 13.02.
Group 1: sham group (sham),
Group 2: CLP group (CLP),
Group 3: compound 26 (10 mg kgꢀ1),
Group 4: compound 26 (20 mg kgꢀ1).
The CLP-induced sepsis model was established aer fasting
2-(4-(4-Fluorophenyl)piperazin-1-yl)-N-(4-(4-uorophenyl)
thiazol-2-yl)acetamide (30). Yield: 83%; mp: 328–329 ꢂC; MW: for 6 h before surgery, under anaesthesia. Aer sterilisation,
414.13; Rf: 0.59; FT-IR (nmax; cmꢀ1 KBr): 3398 (N–H str, –NH), 3145 a 1.5 cm ventral midline abdominal incision was made and the
(Ar C–H str), 2883 (CH2), 2859 (thiazole C–H str), 1718 (C]O str), cecum was then gently isolated and ligated with a 3–0 silk
1647 (C]C str), 1519 (C]N str), 1193 (Ar–F), 658 (C–S str) cmꢀ1
;
suture, punctured with an 18-gauge needle at three locations,
1H-NMR (400 MHz, DMSO, TMS) d ppm: 9.13 (s, 1H, –CONH), 8.15 and then repositioned. The abdomen was then closed. Rats in
(d, 2H, J ¼ 8.8 Hz, Ar–H), 7.54 (s, 1H, thiazole-H), 7.28 (d, 2H, J ¼ the sham group underwent the same surgery, but the cecum
8.2 Hz, Ar–H), 7.04 (d, 2H, J ¼ 8.4 Hz, Ar–H), 6.69 (d, 2H, J ¼ was manipulated without being ligated or perforated. Saline (2
7.5 Hz, Ar–H), 3.46 (dd, 4H, J ¼ 10.2 Hz, piperazine-H), 3.18 (s, 2H, mL/100 g body weight) was given subcutaneously to the rats
CH2), 2.45 (dd, 4H, J ¼ 11.3 Hz, piperazine-H); 13C-NMR (100 immediately aer the operation for resuscitation. For the
MHz, CDCl3) d, ppm: 168.4, 164.3, 162.8, 156.9, 150.3, 145.2, compound 26 treatment groups, rats received 10 or 20 mg kgꢀ1
130.6, 128.7, 116.4, 116.1, 115.9, 105.3, 63.8, 54.3, 51.8; mass: intraperitoneally 30 min before the surgery. Rats in the sham
415.14 (M + 1); elemental analysis for C21H20F2N4OS: calculated: and CLP groups received only the vehicle.
C, 60.85; H, 4.86; N, 13.52; found: C, 60.87; H, 4.86; N, 13.52.
Experimental setup
MMP-2 and MMP-8 inhibitory activity
Setup 1. At 24 h, 24 rats were randomly selected, categorised
Recombinant human pro-MMP-2 and -8 were purchased from into four groups, and used for the assessment of the lung wet/
R&D Systems (Minneapolis, MN, USA), with catalogue numbers dry weight ratio.
32920 | RSC Adv., 2017, 7, 32909–32922
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Setup-2. At 24 h, 24 rats were randomly selected, categorised Immunohistochemistry
RSC Advances
into four groups, and used for histological and molecular bio-
logical examinations. The rats in each group were anaesthetised
and underwent bronchoalveolar lavage in the le lung with 1.5
mL of saline. Aer the bronchoalveolar lavage uid (BALF) and
blood had been collected, the rats were killed, followed by right
lung tissue collection.
Aer being dewaxed and hydrated, sections of lung tissue were
blocked with phosphate-buffered saline (PBS) containing 10%
bovine serum albumin at room temperature and incubated
overnight at 4 ꢂC with cyclooxygenase (COX)-2 (1 : 500, BA0738;
Wanleibio, Shanghai, China) primary antibody. Aer being
incubated in biotinylated goat anti-rabbit secondary antibodies
(1 : 200; Beyotime Institute of Biotechnology) for 30 min,
sections were then incubated with horseradish peroxidase
(HRP)-labelled streptavidin (Beyotime) for 30 min at 37 ꢂC. Each
step was followed by three rinses in PBS. The peroxidase was
visualized by reaction with diaminobenzidine tetrahydro-
chloride. Sections were counterstained with haematoxylin and
analysed under an optical microscope.
Histology
The lung tissues were xed with 4% paraformaldehyde and
dehydrated with a graded concentration of alcohols. Tissues
were xed in paraffin and sliced into 5 mm sections. The
sections were stained with haematoxylin and eosin (H&E) for
3 min and observed under a light microscope.
Protein extraction and western blotting analysis
Measurement of the lung wet/dry weight ratio
Lung tissues were homogenised in NP-40 lysis buffer (Beyotime)
containing 1% Triton X-100 (Sinopharm Chemical Reagent
The rat lungs were weighed immediately aer being harvested
and then dried at 80 ꢂC for 72 h to achieve constant weight. The
wet lung/dry lung ratio was calculated.
Beijing Co., Ltd., Beijing, China) with
1 mM phenyl-
methanesulphonyl uoride (Beyotime Institute of Biotech-
nology) and centrifuged at 12 000g for 10 min. The supernatants
were collected as total protein. Nuclear and cytosolic proteins
were extracted using a nuclear and cytoplasmic protein extrac-
tion kit (Beyotime Institute of Biotechnology) following the
manufacturer's protocol. A commercial bicinchoninic acid
protein assay kit (Beyotime Institute of Biotechnology) was used
for the determination of protein concentration.
For western blot analysis, protein was subjected to 13% (w/v)
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) and electrotransferred onto polyvinylidene
diuoride (PVDF) membranes (Millipore, Billerica, MA, USA).
Aer being blocked with skimmed milk in 1ꢃ PBS in 0.05%
Tween-20 for 1 h, the membrane was incubated overnight with
diluted anti-COX-2 (1 : 1000, BA0738; Boster, Wuhan, China),
inhibitors of NF-kBa (IkBa) (1 : 1000, bs-1287R; Bioss, Beijing,
China), p65 antibodies (1 : 1000, BA0610; Boster), p-p65ser536
antibodies (1 : 1000, bs-0982R; Bioss), Bcl-2 antibodies, and Bax
antibodies in blocking buffer. The blot was washed with 1ꢃ PBS
in 0.05% Tween-20 thrice and incubated with HRP-conjugated
goat anti-rabbit IgG (1 : 5000; Beyotime Institute of Biotech-
nology) for 1 h at room temperature. b-Actin and lamin A were
used as loading controls. The blots were analysed using ImageJ
soware (National Institutes of Health, Bethesda, MD, USA).
The nal data were obtained by normalization with a loading
control and values of the sham group.
Analysis of total protein concentration and cell count of the
BALF
ꢂ
The BALF was centrifuged at 1500 rpm for 10 min at 4 C. The
supernatant was collected for total protein analysis using
a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute
of Biotechnology, Haimen, China), in accordance with the
manufacturer's instructions. The cell pellets were resuspended
and stained with Wright–Giemsa solution for leukocyte count-
ing using a haemocytometer. The cells were classied as
mononuclear and polymorphonuclear based on their normal
morphology.
Biochemical determination in lung tissues
For the measurement of oxidative states in lung tissues, the
activities of myeloperoxidase (MPO) and superoxide dismutase
(SOD) and the levels of malondialdehyde (MDA) and gluta-
thione (GSH) in lung homogenates were measured using cor-
responding commercial kits (Jiancheng Bioengineering
Institute, Nanjing, China). For the measurement of inamma-
tory responses in lung tissues, levels of prostaglandin E2 (PGE2)
were measured using a PGE2 Assay Kit (Wanleibio, Shenyang,
China). All measurements were performed in accordance with
the manufacturer's protocol.
Serum cytokines analysis
Compound 26 treatment of A549 cells (Fig. 7)
The whole blood was centrifuged at 1500 rpm for 10 min at 4 ꢂC A549, a human lung epithelial cell line, was obtained from the
and the serum was collected. Levels of tumour necrosis factor Bioresource Collection and Research Center in Taiwan. Cells
(TNF)-a, interleukin (IL)-1b, and IL-6 in serum were determined were cultured in F-12 nutrient mixture medium (Life Technol-
by enzyme-linked immunosorbent assays using commercial kits ogies, Carlsbad, CA, USA), which contained 10% foetal bovine
specic for rats, in accordance with the manufacturer's serum (Biological Industries, Haemek, Israel), penicillin (100
instructions (USCN Life Science Inc., Wuhan, China). The units per mL), and streptomycin (100 mg mLꢀ1). Compound 26
concentrations are expressed in picograms per milligram was dissolved in DMSO to obtain stock solutions of 10 mg
protein.
mLꢀ1. In all cell experiments, the DMSO concentration in the
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culture medium was #0.1%. A549 cells were pretreated with
compound 26 (1–10 mg mLꢀ1) for 1 h and then stimulated with 1
mg mLꢀ1 LPS for 24 h. The supernatants were collected and
assayed with ELISA kits for the specic detection of selected
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17 M. Bosmann and P. A. Ward, Trends Immunol., 2013, 34(3),
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A549 cells were treated with two doses of compound 26 (10 and
20 mg kgꢀ1) and stimulated with LPS for 24 h, as previously
described (Liou et al., 2016). A calcein-AM solution (Sigma) was
then used to stain acute monocytic leukaemia cells (THP-1
cells). THP-1 cells were co-cultured with the A549 cells to
observe THP-1 adhesion. Adherent cells were detected (green
uorescence) with uorescence microscopy (Olympus, Tokyo,
Japan).
¨ ¨
19 L. Nissinenand and V. M. Kahari, Biochim. Biophys. Acta,
Conclusion
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20 M. Giannandrea and W. C. Parks, Dis. Models & Mech., 2014,
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21 J. Hu, P. E. Van den Steen, Q.-X. a Sang and G. Opdenakker,
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22 E. E. Creemers, J. P. Cleutjens, J. F. Smits and M. J. Daemen,
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We identied a novel series of thiazoles, which exert a potential
protective effect against ALI via the inhibition/modulation of
numerous pathways. This study provides fresh insight that
should aid the development of novel therapeutics against
sepsis-induced ALI; however, a more diverse range of studies are
needed to validate the claims of this study.
23 B. Pirard, Drug Discovery Today, 2007, 12(15–16), 640–646.
24 K. G. Lu and C. M. Stultz, J. Mol. Biol., 2013, 425, 1815–1825.
25 B. J. Rollins, Blood, 1997, 90, 909–928.
Conflict of interest
26 A. Rouf and C. Tanyeli, Eur. J. Med. Chem., 2015, 97, 911–927.
27 Y. Lu, et al., J. Med. Chem., 2009, 52, 1701–1711.
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Authors declare no conict of interest.
Acknowledgements
This study was supported by Jiangsu province Natural Science
Foundation of China (No. BK20151204).
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32922 | RSC Adv., 2017, 7, 32909–32922
This journal is © The Royal Society of Chemistry 2017